U.S. patent application number 10/790060 was filed with the patent office on 2005-03-03 for metal complex-protein composite and hydrogenation catalyst.
This patent application is currently assigned to Nagoya Industrial Science Research Institute. Invention is credited to Abe, Satoshi, Ueno, Takafumi, Watanabe, Yoshihito.
Application Number | 20050049405 10/790060 |
Document ID | / |
Family ID | 34214208 |
Filed Date | 2005-03-03 |
United States Patent
Application |
20050049405 |
Kind Code |
A1 |
Watanabe, Yoshihito ; et
al. |
March 3, 2005 |
Metal complex-protein composite and hydrogenation catalyst
Abstract
The metal complex-protein composite of the present invention
includes a protein having a cavity and a metal complex and has a
specific structure that the metal complex is received in the cavity
of the protein. Here the metal complex is prepared by complexation
of a metal ion, which is selected among the group consisting of
rhodium, ruthenium, and palladium, with a ligand. The metal
complex-protein composite of the invention functions as a
hydrogenation catalyst of an olefin in water. The metal
complex-protein composite is thus effectively applied to
hydrogenation of water-soluble substrates and has environmental
advantages over organic solvents.
Inventors: |
Watanabe, Yoshihito;
(Aichi-ken, JP) ; Ueno, Takafumi; (Aichi-ken,
JP) ; Abe, Satoshi; (Aichi-ken, JP) |
Correspondence
Address: |
OLIFF & BERRIDGE, PLC
P.O. BOX 19928
ALEXANDRIA
VA
22320
US
|
Assignee: |
Nagoya Industrial Science Research
Institute
Nagoya-shi
JP
|
Family ID: |
34214208 |
Appl. No.: |
10/790060 |
Filed: |
March 2, 2004 |
Current U.S.
Class: |
530/400 |
Current CPC
Class: |
C07K 14/805 20130101;
B01J 31/003 20130101 |
Class at
Publication: |
530/400 |
International
Class: |
C07K 014/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 2, 2003 |
JP |
2003-310085 |
Claims
What is claimed is:
1. A metal complex-protein composite, comprising a protein having a
cavity therein and a metal complex prepared by complexation of a
metal ion, which is selected among the group consisting of rhodium,
ruthenium, and palladium, with a ligand, said metal complex-protein
composite having a specific structure that the metal complex is
received in the cavity of the protein.
2. A metal complex-protein composite in accordance with claim 1,
wherein the protein is any one of proteins having either of an
amino acid residue that coordinates to the selected metal ion of
the metal complex and an amino acid residue that forms a
non-covalent bond to the ligand of the metal complex in the cavity
thereof, multimers of such proteins, and variants of such
proteins.
3. A metal complex-protein composite in accordance with claim 1,
wherein the protein is any one of proteins having the cavity in a
heme site by removing a heme from heme-containing proteins,
multimers of such proteins, and variants of such proteins.
4. A metal complex-protein composite in accordance with claim 1,
wherein the protein is selected among the group consisting of
apomyoglobin, apohemoglobin, apoheme oxygenase, apocatalase,
apocytochrome, apoferritin, and their variants.
5. A metal complex-protein composite in accordance with claim 4,
wherein the protein is an apomyoglobin variant having a replacement
of histidine as a 64.sup.th amino acid residue of apomyoglobin.
6. A metal complex-protein composite in accordance with claim 1,
wherein the metal complex is a complex of rhodium with a compound
having a phosphino group as the ligand.
7. A metal complex-protein composite in accordance with claim 6,
wherein the metal complex is a complex of rhodium with a compound
having at least two diphenylphosphino groups as the ligand.
8. A metal complex-protein composite in accordance with claim 6,
wherein the metal complex has the ligand expressed by Formula (1):
R.sup.1R.sup.2P-J-PR.sup.3R.sup.4 (1) where R.sup.1 through R.sup.4
represent any of completely identical, partially identical, and
completely different substituted and non-substituted hydrocarbons
of 1 to 10 carbon atoms and substituted and non-substituted
phenyls, and J represents any of substituted and non-substituted
hydrocarbons of 1 to 10 carbon atoms and two carbon atoms included
in benzene rings.
9. A hydrogenation catalyst, which is a metal complex-protein
composite in accordance with claim 1 and works to accelerate
hydrogenation of an olefin in water.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a novel metal
complex-protein composite and a novel hydrogenation catalyst.
[0003] 2. Description of the Prior Art
[0004] The inventor of the present invention has proposed metal
complex-protein composites of manganese-Schiff base complexes
inserted in a cavity of apomyoglobin (apo-Mb) by non-covalent
bonding. Here apomyoglobin is obtained by liberating a heme from an
oxygen storage protein, myoglobin (Mb). The inventor synthesized,
for example, a metal complex-protein composite including a metal
complex of manganese with
N,N'-bis(salicylidene)-1,2-phenylenediamine kept in the cavity of
apomyoglobin, and reported that such composites were useful for
asymmetric oxidation reaction of thioanisole (the Proceedings of
the 16.sup.th Biofunctional Symposium, `1S1-11 Construction of
Artificial Enzyme by Insertion of Metal Complex into Apomyoglobin
Cavity` (published in September 2001).
[0005] The study of these metal complex-protein composites has just
started, and no useful metal complex-protein composites for
hydrogenation reaction have been reported so far.
SUMMARY OF THE INVENTION
[0006] The object of the invention is thus to provide a novel metal
complex-protein composite. The object of the invention is also to
provide a novel hydrogenation catalyst.
[0007] The inventor of this invention has developed a novel metal
complex-protein composite as a fruit of intensive studies. The
metal complex-protein composite of the present invention includes a
protein having a cavity and a metal complex and has a specific
structure that the metal complex is received in the cavity of the
protein. Here the metal complex is prepared by complexation of a
metal ion, which is selected among the group consisting of rhodium,
ruthenium, and palladium, with a ligand. The metal complex-protein
composite of the invention functions as a hydrogenation catalyst of
an olefin in water. The metal complex-protein composite is thus
effectively applied to hydrogenation of water-soluble substrates
and has environmental advantages over organic solvents.
[0008] Any of diverse methods may be applied to synthesis of the
metal complex-protein composite of the invention. Typically there
are two applicable methods. One method inserts the metal complex
into the cavity of the protein. The other method adds a material of
the metal complex (the material that is changed to the metal
complex by a reaction), which is to be received in the cavity of
the protein, to a system including the protein having the cavity
and synthesizes the metal complex in the system simultaneously with
insertion of the metal complex into the cavity. One concrete
procedure of the former method mixes the protein having the cavity
with the metal complex at an equivalent ratio of 1 to 0.5 through
100 or preferably at an equivalent ratio of 1 to 1.1 through 2.
Preferable solvents for the mixing reaction include mixed solvents
of water and acetone, mixed solvents of water and methanol, mixed
solvents of water and dimethylformamide (DMF), mixed solvents of
water and dimethyl sulfoxide (DMSO), and water alone. Especially
preferable are mixed solvents of water and acetone and mixed
solvents of water and methanol. The mixing temperature is in a
range of -10 to 200.degree. C. and is preferably in a range of 1 to
4.degree. C. The mixing time is in a range of 0.5 minutes to 24
hours and preferably in a range of 5 to 30 minutes. One concrete
procedure of the latter method mixes the protein with the metal ion
at an equivalent ratio of 1 to 0.5 through 100 or preferably at an
equivalent ratio of 1 to 1.1 through 2. Preferable solvents for the
mixing reaction include mixed solvents of water and acetone, mixed
solvents of water and methanol, mixed solvents of water and DMF,
mixed solvents of water and DMSO, and water alone. Especially
preferable are mixed solvents of water and acetone and mixed
solvents of water and methanol. The mixing temperature is in a
range of -10 to 200.degree. C. and is preferably in a range of 1 to
4.degree. C. The mixing time is in a range of 0.5 minutes to 24
hours and preferably in a range of 5 minutes to 1 hour. Another
applicable procedure inserts the metal complex into the cavity of
the protein carried on a carrier by either of the above two
methods. Still another applicable procedure prepares a metal
complex-protein composite and replaces the ligand of the metal
complex with another ligand.
[0009] The protein of the invention may be any one of proteins
having either of an amino acid residue that coordinates to the
selected metal ion of the metal complex and an amino acid residue
that forms a non-covalent bond to the ligand of the metal complex
in the cavity thereof, multimers of such proteins, and variants of
such proteins. The protein of the invention may otherwise be any
one of proteins having the cavity in a heme site by removing a heme
from heme-containing proteins, multimers of such proteins, and
variants of such proteins. Concrete examples include apomyoglobin,
apohemoglobin, apoheme oxygenase, apocatalase, apocytochrome,
apoferritin, and their variants. The terminology `apo` is a prefix
representing a protein having a defective cofactor or a defective
prosthetic group. Apomyoglobin and apohemoglobin have a defective
heme, and apoferritin has a defective iron ion. The variant of the
protein preferably has a replacement of an amino acid residue at a
position affecting the chemical reaction field of the metal complex
received in the cavity of the protein with another amino acid
residue suitable for the chemical reaction. The variant of
apomyoglobin is, for example, apomyoglobin (polypeptide chain of
153 amino acids) having replacement of one or plurality of a
64.sup.th amino acid residue, a 71.sup.st amino acid residue, a
93.sup.rd amino acid residue, and a 107.sup.th amino acid residue.
Especially preferable is an apomyoglobin variant having a
replacement of a 64.sup.th histidine (His64) with an amino acid
residue smaller than histidine, such as glycin or alanine.
[0010] The metal complex of the invention may be any metal complex
of the metal ion coordinating to an amino acid residue located in
the cavity of the protein or any metal complex of the ligand
forming a non-covalent bond to the amino acid residue located in
the cavity of the protein. A metal complex including a compound
having a phosphino group as the ligand is preferable. Especially
preferable is a metal complex including a compound having at least
two diphenylphosphino groups as the ligand. One example of the
preferable ligand is given as Formula (1):
R.sup.1R.sup.2P-J-PR .sup.3R.sup.4 (1)
[0011] where R.sup.1 through R.sup.4 represent any of completely
identical, partially identical, and completely different
substituted and non-substituted hydrocarbons of 1 to 10 carbon
atoms and substituted and non-substituted phenyls, and J represents
any of substituted and non-substituted hydrocarbons of 1 to 10
carbon atoms and two carbon atoms included in benzene rings.
[0012] The phosphino ligand is not specifically restricted, but may
be, for example, any of bis(diphenylphosphino)methane,
bis(diphenylphosphino)ethane, bis(diphenylphosphino)propane,
bis(diphenylphosphino)butane, bis(diphenylphosphino)pentane,
bis(diphenylphosphino)hexane, 1,2-bis(diphenylphosphino)benzene,
bis(dimethylphosphino)methane, bis(dimethylphosphino)ethane,
bis(dimethylphosphino)propane, bis(dimethylphosphino)butane,
bis(dimethylphosphino)pentane, bis(dimethylphosphino)hexane,
1,2-bis(dimethylphosphino)benzene, and bis(diphenylphosphino)
compounds having one or more hydrogen atoms in the phenyl group
displaced by any of substituent groups including alkyl groups,
alkoxy groups, nitro groups, carboxyl groups, and halogens. These
phosphino ligands are preferably used for the ligand of rhodium
complexes and palladium complexes.
[0013] The ligand is not restricted to the phosphino ligand but may
be a cyclic diene or an aromatic compound that reacts with a metal
to produce a metallocene compound. Typical examples of the cyclic
diene include cyclopentadiene, cyclooctadiene, and cyclopentadiene
and cyclooctadiene derivatives having one or more hydrogen atoms
displaced by any of substituent groups including alkyl groups,
alkoxy groups, nitro groups, carboxyl groups, and halogens. Typical
examples of the aromatic compound include benzene, naphthalene, and
benzene and naphthalene derivatives having one or more hydrogen
atoms displaced by any of substituent groups including alkyl
groups, alkoxy groups, nitro groups, and carboxyl groups, for
example, toluene, xylene, isopropyl benzene, isobutyl benzene, o-,
m-, and p-isopropyltoluene (cymene), and o-, m-, and
p-isobutyltoluene. These ligands are preferably used for the ligand
of ruthenium complexes.
[0014] The hydrogenation catalyst of the invention is composed of
the metal complex-protein composite discussed above and functions
to accelerate hydrogenation in water. The amount of the
hydrogenation catalyst used depends upon the reaction vessel and
the economical efficiency. The molar ratio S/C (where S denotes a
reaction substrate and C denotes the catalyst) is preferably in a
range of 10 to 10000 or more specifically in a range of 50 to 5000.
The reaction substrate is not specifically restricted but may be
any compound having a site to be hydrogenated. The reaction
substrate is preferably water-soluble, since hydrogenation takes
place in water. Any aqueous solvent may be used for the solvent of
the hydrogenation reaction. Typical examples include water, mixed
solvents of water and lower alcohols (for example, methanol and
ethanol), mixed solvents of water and lower ketones (for example,
acetone and methyl ethyl ketone), mixed solvents of water and DMF,
and mixed solvents of water and DMSO. The reaction temperature is
in a range of -10 to 200.degree. C. and is preferably in a range of
1 to 50.degree. C. The mixing time is in a range of 0.5 minutes to
24 hours and is preferably in a range of 5 minutes to 10 hours.
This hydrogenation reaction may be in a batchwise operation or in a
flow operation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows Examples 3 through 6; and
[0016] FIG. 2 shows syntheses of various metal complex-protein
composites.
EXAMPLES
[0017] Some examples of the invention are discussed below. In the
description below, `cod`, `dppe`, and `dppb` respectively represent
1,5-cyclooctadiene, 1,2-bis(diphenylphosphino)ethane, and
1,4-bis(diphenylphosphino)butane.
Example 1
Synthesis of Rhodium Complex 1
[0018] Rh(I)(cod)(dppe) was synthesized according to the procedure
disclosed in a cited reference (Brown, J. M. et al., Journal of
Organometallic Chemistry, 1981, vol216, p263-276). The procedure of
synthesis mixed [Rh(cod)Cl].sub.2 (99 mg, 0.2 mmol) with AgBF.sub.4
(80 mg, 0.41 mmol) in acetone in an atmosphere of argon with
stirring for three hours and added solid dppe (159 mg, 0.4 mmol) to
yield a red solution. The procedure concentrated the red
supernatant to 3 ml and added ether (20 ml) to the concentrate to
yield a yellow precipitate. The yellow precipitate was washed with
ether and was evaporated. This gave an object compound,
Rh(I)(cod)(dppe).BF4. The observed values by ESI-TOF MS
(electrospray ionization time-of-flight mass spectrometry) were
[Rh(I)(cod)(dppe)].sup.+m/z: 609.10 (calculated value: 609.14),
[Rh(I)(dppe)(CH.sub.3OH)].sup.+m/z: 533.03 (calculated value:
533.38), and [Rh(dppe)].sup.+m/z] 501.02 (calculated value:
501.04).
Example 2
Synthesis of Rhodium Complex 2
[0019] Rh(I)(cod)(dppb) was purchased from Sigma-Aldrich Inc. The
product name was [1,4-bis(diphenylphosphino)butane]
(1,5-cyclooctadiene)rhodium tetrafluoroborate.
Example 3
Synthesis of Rhodium Complex-Apomyoglobin Composite 1 (see FIG.
1)
[0020] All the operations for the synthesis were performed at a
temperature of 4.degree. C. Histidine as a 64.sup.th amino acid
residue of myoglobin was replaced with alanine according to the
procedure disclosed in a cited reference (T. Matsui et al. J. Am.
Chem. Soc., 1999, vol121, p9952-9957). The variant myoglobin is
hereafter referred to as SW H64A Mb. The variant myoglobin SW H64A
Mb was processed by the acid-butanone method described in a cited
reference (F. Ascole et al. Method Enzymol. 1981, vol76, p72-87)
and was successively dialyzed with 1 mM, 5 mM, and 10 mM Tris/HCl
buffer solutions (pH 7.0) for 2 hours each. This gave apomyoglobin,
which is hereafter referred to as apo-H64A Mb. The procedure then
mixed apo-H64A Mb with 10 mM Tris/HCl buffer solution (pH 7.0) (385
.mu.M, 18 ml), added the acetone solution of the rhodium complex
(10 mM, 1.038 ml) obtained in Example 1 with stirring to an
equivalent ratio of 1.5 Rh to 1 Mb, and stood still at 4.degree. C.
for 10 minutes. The resulting mixed solution was dialyzed overnight
with 1 liter of 10 mM Bis Tris/HCl buffer solution (pH 6.0). The
reconstructed rhodium complex-apomyoglobin composite
Rh(dppe)-apo-H64A Mb was purified by gel filtration with G25 and
G50 (10 mM Tris/HCl buffer solution (pH7.0)). Here G25 and G50
respectively represent Sephadex G25 Medium and Sephadex G50 Medium
(manufactured by Amersham Biosciences K.K.). The resulting
composite was identified by ESI-TOF MS, UV-vis analysis, and atomic
absorption spectroscopy. The observed value by ESI-TOF MS was
17764.8, which well agreed with the calculated value 17765.4. The
absorption maximum wavelength of the composite in UV-vis
(ultraviolet-visible spectroscopy) was 259.5 nm, which was lower
than the absorption maximum wavelength of apo-H64A Mb (280 nm). The
concentration of Rh was determined to be 1.77 mM by atomic
absorption spectroscopy.
Example 4
Synthesis of Rhodium Complex-Apomyoglobin Composite 2 (see FIG.
1)
[0021] A rhodium complex-apomyoglobin composite
Rh(dppe).multidot.apo-Mb was obtained according to the same
procedure as that of Example 3, except that myoglobin was not
replaced. The observed value of the resulting composite by ESI-TOF
MS was 17829.9, which well agreed with the calculated value
17831.1. The absorption maximum wavelength of the composite in
UV-vis analysis was 274.5 nm, which was lower than the absorption
maximum wavelength of apo-Mb (280 nm). The concentration of Rh was
determined to be 1.13 mM by atomic absorption spectroscopy.
Example 5
Synthesis of Rhodium Complex-Apomyoglobin Composite 3 (see FIG.
1)
[0022] A rhodium complex-apomyoglobin composite
Rh(dppb).multidot.apo-Mb was obtained according to the same
procedure as that of Example 4, except that the rhodium complex
obtained in Example 2 was used instead of the rhodium complex
obtained in Example 1. The observed value of the resulting
composite by ESI-TOF MS was 17859.8, which well agreed with the
calculated value 17859.2.
Example 6
Synthesis of Rhodium Complex-Apomyoglobin Composite 4 (see FIG.
1)
[0023] Another method was applied to synthesize a rhodium
complex-apomyoglobin composite. This method synthesizes a rhodium
complex in situ in the presence of apomyoglobin to obtain the
rhodium complex-apomyoglobin composite. The procedure added an
acetone solution of [Rh(cod)Cl].sub.2 (2 mM, 2 .mu.l) (at an
equivalent ratio of 2 Rh to 1 Mb) and an acetone solution of dppe
(2 mM, 4 .mu.l) to a 5 mM ammonium acetate solution of apo-H64D Mb
(having a replacement of a 64.sup.th histidine with aspartic acid)
(20 .mu.M, 200 .mu.l) and stood the mixed solution still at
4.degree. C. for 1 hour. The observed value of the resulting
composite by ESI-TOF MS was 17808.0, which well agreed with the
calculated value 17809.1 of the composite of cod-free Rh(I)(dppe)
and apo-H64D Mb.
Example 7
Hydrogenation Reaction of Olefin 1
[0024] The rhodium complex-apomyoglobin composite
Rh(dppe).multidot.apo-H6- 4A Mb obtained in Example 3 was used for
hydrogenation reaction of acrylic acid. The concentration of Rh of
the purified composite was determined by atomic absorption
spectroscopy. Acrylic acid went through a hydrogenation reaction in
50 mM phosphate buffer (pD 7.0) for 5 hours under the conditions of
[Rh]/[substrate]=1/100, a temperature of 35.degree. C., and a
hydrogen pressure of 5 atm. Here pD represents -log.sub.10[D+] (D
is deuterium). The procedure placed an aqueous solution of the
rhodium complex-apomyoglobin composite (0.5 mM, 1 ml, 0.5 .mu.mol)
in an auto clave, added an aqueous solution of acrylic acid (50 mM,
1 ml, 50 .mu.mol) to the aqueous solution of the rhodium
complex-apomyoglobin composite, and replaced the atmosphere in the
auto clave with gaseous hydrogen for the hydrogenation reaction
under the above conditions. This hydrogenation reaction changed
acrylic acid to propionic acid. The turnover number measured by
.sup.1H-NMR was 0.68 h.sup.-1.
Example 8
Hydrogenation Reaction of Olefin 2
[0025] Acrylamide was subjected to hydrogenation reaction with the
rhodium complex-apomyoglobin composite Rh(dppe).multidot.apo-Mb
obtained in Example 4, according to the same procedure as that of
Example 7. This hydrogenation reaction changed acrylamide to
propionamide. The turnover number measured by .sup.1H-NMR was 0.60
h.sup.-1.
Example 9
[0026] Various metal complex-protein composites were synthesized
according to reaction formulae shown in FIGS. 2(a) through 2(d).
Apomyoglobin used here was apo-H64D Mb. The observed values of the
resulting metal complex-protein composites by ESI-TOF MS were also
shown in FIG. 2. Concrete procedures of the syntheses were
discussed below.
[0027] FIG. 2(a): The procedure of the synthesis mixed an apo-H64D
solution (212 .mu.M, 14 ml) with an acetone solution of
[Rh(cod)Cl].sub.2 (10 mM, 150 .mu.l) and stood the mixed solution
still at 4.degree. C. for 10 minutes. The mixed solution was
dialyzed overnight with 10 mM Bis Tris/HCl buffer solution (pH
6.0). The reconstructed rhodium complex-apomyoglobin composite
Rh(cod).multidot.apo-H64D Mb was purified by gel filtration with
G25 and G50. The observed value of the composite by ESI-TOF MS was
17516.6, which well agreed with the calculated value 17519.1.
[0028] FIG. 2(b): A palladium complex-apomyoglobin composite
Pd(dppe)-apo-H64D Mb was obtained according to the same procedure
as that of FIG. 2(a), except that a Pd(dppe)DMF solution prepared
by mixing Pd(dppe)Cl.sub.2 and AgBF.sub.4 in DMF and liberating Cl
was used as the metal complex solution. The observed value of the
composite by ESI-TOF MS was 17814.8, which well agreed with the
calculated value 17812.0.
[0029] FIG. 2(c): A methanol solution of
dichloro(p-cymene)ruthenium dimmer (2 equivalent weight) was added
to a 5 mM ammonium acetate solution of apo-H64D Mb (20 .mu.M, 200
.mu.l), and the mixed solution was stood still at 4.degree. C. for
1 hour. The observed value of the composite by ESI-TOF MS was
17544.3, which well agreed with the calculated value 17543.4.
[0030] FIG. 2(d): A ruthenium complex-apomyoglobin composite was
obtained according to the same procedure as that of FIG. 2(c),
except that a mixture of a methanol solution of
dichloro(p-cymene)ruthenium dimmer (10 mM, 100 .mu.l) and a
methanol solution of 1,8-diaminonaphthalene (20 mM, 100 .mu.l)
mixed at room temperature, stirred for 1 minute, and stood still
overnight at room temperature was used as the metal complex
solution. The observed value of the composite by ESI-TOF MS was
17700.9, which well agreed with the calculated value 17699.6.
[0031] A composite of apocytochrome c was discussed as another
example. The procedure mixed apocytochrome c with ruthenium
chloride (p-cymene)(4-methyl-1,2-benzenediamine) at a rate of 1 to
1 or 1 to 2 and placed the mixture on ice for more than 10 minutes.
The mixture was dialyzed with ammonium acetate buffer (5 mM, pH
6.8, 4.degree. C.) for 12 hours and was passed through a G50 gel
filtration column equilibrated by ammonium acetate buffer (5 mM, pH
6.8 4.degree. C.) for purification. This gave a ruthenium
(p-cymene)(4-methyl-1,2-benzenediamine)-apocytochro- me c
composite. The observed value of the composite by ESI-TOF MS was
12097.1, which well agreed with the calculated value 12098.
* * * * *